poly(ADP-ribose) Polymerase Overactivation Research Articles

Cytoprotective effect of gallotannin in oxidatively stressed HaCaT keratinocytes: the role of poly(ADP-ribose) metabolism.

Oxidative stress-induced cytotoxicity is mediated in part by accelerated poly-ADP ribosylation. Peroxynitrite and hydrogen peroxide cause DNA breakage triggering the activation of the DNA nick sensor enzyme poly(ADP-ribose) polymerase-1 (PARP-1). Overactivation of PARP-1 leads to cell dysfunction and cell death mainly due to depletion of NAD(+) (the substrate of PARP-1) and ATP. PARP-1 attaches most ADP-ribose residues onto itself, leading to downregulation of enzyme activity. Here, we have investigated the role of poly(ADP-ribose) glycohydrolase (PARG), the poly(ADP-ribose)-catabolyzing enzyme in oxidative stress-induced cytotoxicity in HaCaT cells. We have found that inhibition of PARG by gallotannin (GT) (50 microM) provided significant cytoprotection to peroxynitrite- or hydrogen peroxide-treated HaCaT cells, as assessed by lactate dehydrogenase release and propidium iodide uptake (parameters of necrotic cell death) as well as caspase activation (apoptotic parameter). GT pretreatment has also inhibited the depletion of cellular NAD(+) pools in hydrogen peroxide- or peroxynitrite-treated HaCaT cells. GT caused the accumulation of poly(ADP-ribose) and concomitant inhibition in cellular PARP activity in oxidatively stressed cells. Therefore, PARG is likely to contribute to maintaining the active state of PARP-1 by continuously removing inhibitory ADP-ribose residues from PARP-1.

Transcription regulation of TNF-alpha-early response genes by poly(ADP-ribose) polymerase-1 in murine heart endothelial cells.

Poly(ADP-ribose) polymerase-1 (PARP-1) has been involved in endothelial cell dysfunction associated with various pathophysiological conditions. The intrinsic mechanism of PARP-1-mediated endothelial cell dysfunction could be related to PARP-1 overactivation, NAD(+) consumption and ATP depletion. An alternative way could involve transcription regulation. By using high-density microarrays, we examined early tumor necrosis factor alpha (TNF-alpha)-stimulated gene expression profiles in PARP-1(+/+) and PARP-1(-/-) murine heart endothelial cells. TNF-alpha modulated a significant number of genes in both cell types. We have identified a set of genes whose expression in response to TNF-alpha is modulated by PARP-1, whereas the expression of others is PARP-1-independent. Up-regulation of several genes involved in the inflammatory response is hampered in the absence of PARP-1. Moreover, NF-kappaB-dependent transcriptional activation is partially inhibited in PARP-1(-/-) compared to PARP-1(+/+) cells. However, we found that PARP-1 might also silence transcription of several NF-kappaB target genes. Overall, our results show that PARP-1 is regulating the expression of genes by the endothelial cells both in a positive and a negative fashion, with the final effects depending on the gene. Individual studies of these genes are now necessary to clarify the intrinsic mechanism by which PARP-1 is controlling transcription and thereby finding out different therapeutic approaches involving PARP-1.

The PARP inhibitor benzamide protects against kainate and NMDA but not AMPA lesioning of the mouse striatum in vivo

Overactivation of poly(ADP-ribose) polymerase (PARP) in response to genotoxic insults can cause cell death by energy deprivation. We previously reported that neurotoxic amounts of kainic acid (KA) injected into the rat striatum produce time-dependent changes in striatal PARP activity in vivo. Here, we have investigated the time-course of KA-induced toxicity and the effects of the PARP inhibitor benzamide on KA, AMPA and NMDA neurotoxicities in vivo, by measuring changes in the volume of the lesion and in NAD+ and ATP levels induced by the intra-striatal injection of these excitotoxins in C57Bl/6N mice. The KA-induced lesion volume was dependent on the amount of toxin injected and the survival time. The lesion was well developed at 48 h and was almost undetectable after one week. KA produced an extensive astrogliosis at one week. Benzamide partially prevented both KA- and NMDA- but not AMPA-induced lesions when measured at 48 h after the treatment. The effects of benzamide appeared to be in part related to changes in energy metabolism, since KA produced decreases in striatal levels of NAD+ and ATP that were partially prevented by benzamide at 48 h and which returned to control levels at one week. NMDA did not affect NAD+ and induced little alteration in ATP levels. Benzamide had no effect on AMPA-induced decreases in either NAD+ or ATP levels at 48 h. These results (1) indicate that PARP overactivation and energy depletion could be responsible in part for the cellular demise during the development of the lesion induced by KA; (2) confirm that PARP is involved in NMDA but not AMPA toxicities; (3) suggest the existence of differences between KA and AMPA-mediated toxicities; and (4) provide further evidence supporting PARP as a novel target for new drug treatments against neurodegenerative disorders.

Poly(ADP-ribose) polymerase inhibition prevents both apoptotic-like delayed neuronal death and necrosis after H(2)O(2) injury.

Toxic reactive oxygen species (ROS) such as hydrogen peroxide, nitric oxide, superoxide, and the hydroxyl radical are generated in a variety of neuropathological conditions and cause significant DNA damage. We determined the effects of 3-aminobenzamide (AB), an inhibitor of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), on cell death in differentiated PC12 cells, a model of sympathetic neurons, after H(2) O(2) injury. Exposure to 0.5 mm H(2) O(2) resulted in a significant decrease in intracellular NAD(H), NADP(H), and ATP levels. This injury resulted in the death of 90% of the cells with significant necrosis early (2 h) after injury and increased apoptosis (12-24 h after injury), as measured by PS exposure and the presence of cytoplasmic oligonucleosomal fragments. Treatment with 2.5 mm AB restored pyridine nucleotide and ATP levels and ameliorated cell death (65% versus 90%) by decreasing the extent of both necrosis and apoptosis. Interestingly, we observed that H(2) O(2) -induced injury caused a delayed cell death exhibiting features of apoptosis but in which caspase-3 like activity was absent. Moreover, pretreatment with AB restored caspase-3-like activity. Our results suggest that apoptosis and necrosis are both triggered by PARP overactivation, and that maintenance of cellular energy levels after injury by inhibiting PARP shifts cell death from necrosis to apoptosis.

Nitric oxide-peroxynitrite-poly(ADP-ribose) polymerase pathway in the skin.

In the last decade it has become well established that in the skin, nitric oxide (NO), a diffusable gas, mediates various physiologic functions ranging from the regulation of cutaneous blood flow to melanogenesis. If produced in excess, NO combines with superoxide anion to form peroxynitrite (ONOO-), a cytotoxic oxidant that has been made responsible for tissue injury during shock, inflammation and ischemia-reperfusion. The opposite effects of NO and ONOO- on various cellular processes may explain the 'double-edged sword' nature of NO depending on whether or not cellular conditions favour peroxynitrite formation. Peroxynitrite has been shown to activate the nuclear nick sensor enzyme, poly(ADP-ribose) polymerase (PARP). Overactivation of PARP depletes the cellular stores of NAD+, the substrate of PARP, and the ensuing 'cellular energetic catastrophy' results in necrotic cell death. Whereas the role of NO in numerous skin diseases including wound healing, burn injury, psoriasis, irritant and allergic contact dermatitis, ultraviolet (UV) light-induced sunburn erythema and the control of skin infections has been extensively documented, the intracutaneous role of peroxynitrite and PARP has not been fully explored. We have recently demonstrated peroxynitrite production, DNA breakage and PARP activation in a murine model of contact hypersensitivity, and propose that the peroxynitrite-PARP route represents a common pathway in the pathomechanism of inflammatory skin diseases. Here we briefly review the role of NO in skin pathology and focus on the possible roles played by peroxynitrite and PARP in various skin diseases.

Reduction of acute photodamage in skin by topical application of a novel PARP inhibitor

The ultraviolet (UV) components of sunlight induce damage to the DNA in skin cells, which is considered to be the initiating step in the harmful biological effects of UV radiation. Repair of DNA damage results in the formation of single-strand DNA breaks, which activate the nuclear poly(ADP-ribose) polymerase (PARP). Overactivation of PARP worsens the oxidative cell damage and impairs the energy metabolism, raising the possibility that moderation of PARP activation following DNA damage may protect skin cells from UV radiation. The topical effects of the novel PARP inhibitor O-(3-pyperidino-2-hydroxy-1-propyl) pyridine-3-carboxylic acid amidoxime monohydrochloride (BGP-15M) were investigated on UV-induced skin damage in a hairless mouse model. For evaluation of the UV-induced acute photodamage to the skin and the potential protective effect of BGP-15M, DNA injury was detected by measuring the formation of single-strand DNA breaks and counting the resulting sunburn (apoptotic) cells. The ADP-ribosylation of PARP was assessed by Western blot analysis and then quantified. In addition, the UV-induced immunosuppression was investigated by the immunostaining of tumor necrosis factor alpha and interleukin-10 expressions in epidermal cells. The signs of inflammation were examined clinically and histochemically. Besides its primary effect in decreasing the activity of nuclear PARP, topically applied BGP-15M proved to be protective against solar and artificial UV radiation-induced acute skin damage. The DNA injury was decreased ( P<0.01). An inhibition of immunosuppression was observed by down-regulation of the epidermal production of cytokines IL-10 and TNFα. In the mouse skin, clinical or histological signs of UV-induced inflammation could not be observed. These data suggest that BGP-15M directly interferes with UV-induced cellular processes and modifies the activity of PARP. The effects provided by topical application of the new PARP-regulator BGP-15M indicate that it may be a novel type of agent in photoprotection of the skin.

Detection of poly(ADP-ribose) polymerase activation in oxidatively stressed cells and tissues using biotinylated NAD substrate.

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme activated by DNA damage. Activated PARP cleaves NAD(+) into nicotinamide and (ADP-ribose) and polymerizes the latter on nuclear acceptor proteins. Over-activation of PARP by reactive oxygen and nitrogen intermediates represents a pathogenetic factor in various forms of inflammation, shock, and reperfusion injury. Using a novel commercially available substrate, 6-biotin-17-nicotinamide-adenine-dinucleotide (bio-NAD(+)), we have developed three applications, enzyme cytochemistry, enzyme histochemistry, and cell ELISA, to detect the activation of PARP in oxidatively stressed cells and tissues. With the novel assay we were able to detect basal and hydrogen peroxide-induced PARP activity in J774 macrophages. We also observed that mitotic cells display remarkably elevated PARP activity. Hydrogen peroxide-induced PARP activation could also be detected in wild-type peritoneal macrophages but not in macrophages from PARP-deficient mice. Application of hydrogen peroxide to the skin of mice also induced bio-NAD(+) incorporation in the keratinocyte nuclei. Hydrogen peroxide-induced PARP activation and its inhibition by pharmacological PARP inhibitors could be detected in J774 cells with the ELISA assay that showed good correlation with the traditional [(3)H]-NAD incorporation method. The bio-NAD(+) assays represent sensitive, specific, and non-radioactive alternatives for detection of PARP activation.

Gain-of-function of poly(ADP-ribose) polymerase-1 upon cleavage by apoptotic proteases: implications for apoptosis.

Poly(ADP-ribosyl)ation is an important mechanism for the maintenance of genomic integrity in response to DNA damage. The enzyme responsible for poly(ADP-ribose) synthesis, poly(ADP-ribose) polymerase 1 (PARP-1), has been implicated in two distinct modes of cell death induced by DNA damage, namely apoptosis and necrosis. During the execution phase of apoptosis, PARP-1 is specifically proteolyzed by caspases to produce an N-terminal DNA-binding domain (DBD) and a C-terminal catalytic fragment. The functional consequence of this proteolytic event is not known. However, it has recently been shown that overactivation of full-length PARP-1 can result in energy depletion and necrosis in dying cells. Here, we investigate the molecular basis for the differential involvement of PARP-1 in these two types of cellular demise. We show that the C-terminal apoptotic fragment of PARP-1 loses its DNA-dependent catalytic activity upon cleavage with caspase 3. However, the N-terminal apoptotic fragment, retains a strong DNA-binding activity and totally inhibits the catalytic activity of uncleaved PARP-1. This dominant-negative behavior was confirmed and extended in cellular extracts where DNA repair was completely inhibited by nanomolar concentrations of the N-terminal fragment. Furthermore, overexpression of the apoptotic DBD in mouse fibroblast inhibits endogenous PARP-1 activity very efficiently in vivo, thereby confirming our biochemical observations. Taken together, these experiments indicate that the apoptotic DBD of PARP-1 acts cooperatively with the proteolytic inactivation of the enzyme to trans-inhibit NAD hydrolysis and to maintain the energy levels of the cell. These results are consistent with a model in which cleavage of PARP-1 promotes apoptosis by preventing DNA repair-induced survival and by blocking energy depletion-induced necrosis.

Poly(ADP-ribose) polymerase inhibitors attenuate necrotic but not apoptotic neuronal death in experimental models of cerebral ischemia.

An excessive activation of poly(ADP-ribose) polymerase (PARP) has been proposed to play a key role in post-ischemic neuronal death. We examined the neuroprotective effects of the PARP inhibitors benzamide, 6(5H)-phenanthridinone, and 3,4-dihydro-5-[4-1(1-piperidinyl)buthoxy]-1(2H)-isoquinolinone in three rodent models of cerebral ischemia. Increasing concentrations of the three PARP inhibitors attenuated neuronal injury induced by 60 min oxygen-glucose deprivation (OGD) in mixed cortical cell cultures, but were unable to reduce CA1 pyramidal cell loss in organotypic hippocampal slices exposed to 30 min OGD or in gerbils following 5 min bilateral carotid occlusion. We then examined the necrotic and apoptotic features of OGD-induced neurodegeneration in cortical cells and hippocampal slices using biochemical and morphological approaches. Cortical cells exposed to OGD released lactate dehydrogenase into the medium and displayed ultrastructural features of necrotic cell death, whereas no caspase-3 activation nor morphological characteristics of apoptosis were observed at any time point after OGD. In contrast, a marked increase in caspase-3 activity was observed in organotypic hippocampal slices after OGD, together with fluorescence and electron microscope evidence of apoptotic neuronal death in the CA1 subregion. Moreover, the caspase inhibitor Z-VAD-FMK reduced OGD-induced CA1 pyramidal cell loss. These findings suggest that PARP overactivation may be an important mechanism leading to post-ischemic neurodegeneration of the necrotic but not of the apoptotic type.

Sulfur Mustard-Induced Apoptosis in Hairless Guinea Pig Skin

Sulfur mustard [bis-(2-chloroethyl)sulfide; HD] causes incapacitating injuries to the eyes, respiratory tract, and skin. Despite decades of research, the mechanism of toxic action of HD is still poorly understood. One proposed toxicological mechanism that triggers cell death involves DNA damage induced by HD alkylation. According to this hypothesis, HD-induced DNA damage activates NAD+, requiring the enzyme poly(ADP-ribose) polymerase (PARP) to initiate DNA repair processes. Overactivation of PARP leads to depletion of NAD+, and, in efforts to resynthesize NAD+, cellular ATP is depleted, and the cell dies from intracellular energy. However, recent studies only partial support this hypothesis. Niacinamide, a substrate for NAD+ synthesis, prevented NAD+ depletion, but did not significantly prevent cell death following HD exposure. These studies suggest cell death occurs independently of depletion of cellular NAD+ with some other pathogenic processes operating in cell death following HD cytotoxicity.The present study examined the role of apoptosis in basal cell degeneration caused by HD exposure in the hairless guinea pig.

Purines inhibit poly(ADP‐ribose) polymerase activation and modulate oxidant‐induced cell death

Purines such as adenosine, inosine, and hypoxanthine are known to have potent antiinflammatory effects. These effects generally are believed to be mediated by cell surface adenosine receptors. Here we provide evidence that purines protect against oxidant-induced cell injury by inhibiting the activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP). Upon binding to broken DNA, PARP cleaves NAD+ into nicotinamide and ADP-ribose and polymerizes the latter on nuclear acceptor proteins such as histones and PARP itself. Overactivation of PARP depletes cellular NAD+ and ATP stores and causes necrotic cell death. We have identified some purines (hypoxanthine, inosine, and adenosine) as potential endogenous PARP inhibitors. We have found that purines (hypoxanthine > inosine > adenosine) dose-dependently inhibited PARP activation in peroxynitrite-treated macrophages and also inhibited the activity of the purified PARP enzyme. Consistently with their PARP inhibitory effects, the purines also protected interferon gamma + endotoxin (IFN/LPS) -stimulated RAW macrophages from the inhibition of mitochondrial respiration and inhibited nitrite production from IFN/LPS-stimulated macrophages. We have selected hypoxanthine as the most potent cytoprotective agent and PARP inhibitor among the three purine compounds, and investigated the mechanism of its cytoprotective effect. We have found that hypoxanthine protects thymocytes from death induced by the cytotoxic oxidant peroxynitrite. In line with the PARP inhibitory effect of purines, hypoxanthine has prevented necrotic cell death while increasing caspase activity and DNA fragmentation. As previously shown with other PARP inhibitors, hypoxanthine acted proximal to mitochondrial alterations as hypoxanthine inhibited the peroxynitrite-induced mitochondrial depolarization and secondary superoxide production. Our data imply that purines may serve as endogenous PARP inhibitors. We propose that, by affecting PARP activation, purines may modulate the pattern of cell death during shock, inflammation, and reperfusion injury.

Poly(ADP-ribosyl)ation basally activated by DNA strand breaks reflects glutamate-nitric oxide neurotransmission.

Poly(ADP-ribose) polymerase (PARP) transfers ADP ribose groups from NAD(+) to nuclear proteins after activation by DNA strand breaks. PARP overactivation by massive DNA damage causes cell death via NAD(+) and ATP depletion. Heretofore, PARP has been thought to be inactive under basal physiologic conditions. We now report high basal levels of PARP activity and DNA strand breaks in discrete neuronal populations of the brain, in ventricular ependymal and subependymal cells and in peripheral tissues. In some peripheral tissues, such as skeletal muscle, spleen, heart, and kidney, PARP activity is reduced only partially in mice with PARP-1 gene deletion (PARP-1(-/-)), implicating activity of alternative forms of PARP. Glutamate neurotransmission involving N-methyl-d-aspartate (NMDA) receptors and neuronal nitric oxide synthase (nNOS) activity in part mediates neuronal DNA strand breaks and PARP activity, which are diminished by NMDA antagonists and NOS inhibitors and also diminished in mice with targeted deletion of nNOS gene (nNOS(-/-)). An increase in NAD(+) levels after treatment with NMDA antagonists or NOS inhibitors, as well as in nNOS(-/-) mice, indicates that basal glutamate-PARP activity regulates neuronal energy dynamics.

Poly(ADP-ribose) polymerase is a mediator of necrotic cell death by ATP depletion.

Apoptotic and necrotic cell death are well characterized and are influenced by intracellular ATP levels. Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated by DNA strand breaks, physiologically participates in DNA repair. Overactivation of PARP after cellular insults can lead to cell death caused by depletion of the enzyme's substrate beta-nicotinamide adenine dinucleotide and of ATP. In this study, we have differentially elicited apoptosis or necrosis in mouse fibroblasts. Fibroblasts from PARP-deficient (PARP(-/-)) mice are protected from necrotic cell death and ATP depletion but not from apoptotic death. These findings, together with cell death patterns in PARP(-/-) animals receiving other types of insults, indicate that PARP activation is an active trigger of necrosis, whereas other mechanisms mediate apoptosis.